The emerging concept of vascular normalization has changed the paradigm of antiangiogenic treatment in cancer (Jain, Nature Medicine, 2001;Jain, Science, 2005;Willett, Nature Medicine, 2004) and raised many critical, yet unanswered, questions concerning tumor vessel biology. The studies in this Project address two critical questions: 1) how do antiangiogenic agents currently in clinical trials affect delivery of chemotherapeutic agents and 2) which surrogate markers can be used to identify the normalization of the tumor vasculature. To answer these questions we will use our pre-clinical models: breast tumor (MCalV, Tong, Cancer Research, 2004) in a mammary fat pad chamber, and glioma (U87MG, Winkler, Cancer Cell, 2004) in a cranial window. In Aim 1,we will compare the normalization of blood vessels after treatment with anti-VEGF antibody (B20-4.1, the mouse equivalent of bevacizumab), anti-VEGFR2 antibody (DC101), or three tyrosine kinase inhibitors, all currently in clinical trials. Specifically,we will quantify the enhancement of transvascular pressure gradients and uniformity of drug delivery. In Aim 2, we will examine vascular basement membrane degradation during vascular normalization by assessing matrix metalloproteinase and tissue inhibitor metalloproteinase levels and their activities in tissue. We will examine the potential ofmatrix metalloproteinases and their degradation products as blood-borne surrogate .markers for vascular normalization. In Aim 3, we will consider the fate of "excess" endothelial cells from tumor vessels pruned or reduced in diameter by antiangiogenic therapy. We hypothesize that many of these cells enter the blood stream and are non-viable. We propose to detect them in peripheral blood and use them as a surrogate marker to evaluate vascular normalization during antiangiogenic therapy. The data obtained in this project will complement those obtained in Project 1 and will facilitate rapid translation into the clinic. If one surrogate marker evaluated in this Project is successful, it will help to advance the combined use of antiangiogenic and cytotoxic therapies.